The impact of methodology on the reproducibility and rigor of DNA methylation data

Epigenetic modifications are crucial for normal development and implicated in disease pathogenesis. While epigenetics continues to be a burgeoning research area in neuroscience, unaddressed issues related to data reproducibility across laboratories remain. Separating meaningful experimental changes from background variability is a challenge in epigenomic studies. Here we show that seemingly minor experimental variations, even under normal baseline conditions, can have a significant impact on epigenome outcome measures and data interpretation. We examined genome-wide DNA methylation and gene expression profiles of hippocampal tissues from wild-type rats housed in three independent laboratories using nearly identical conditions. Reduced-representation bisulfite sequencing and RNA-seq respectively identified 3852 differentially methylated and 1075 differentially expressed genes between laboratories, even in the absence of experimental intervention. Difficult-to-match factors such as animal vendors and a subset of husbandry and tissue extraction procedures produced quantifiable variations between wild-type animals across the three laboratories. Our study demonstrates that seemingly minor experimental variations, even under normal baseline conditions, can have a significant impact on epigenome outcome measures and data interpretation. This is particularly meaningful for neurological studies in animal models, in which baseline parameters between experimental groups are difficult to control. To enhance scientific rigor, we conclude that strict adherence to protocols is necessary for the execution and interpretation of epigenetic studies and that protocol-sensitive epigenetic changes, amongst naive animals, may confound experimental results

A Boost in the Paycheck: Survey Evidence on Workers’ Response to the 2011 Payroll Tax Cuts

This paper presents new survey evidence on workers' response to the 2011 payroll tax cuts. While workers intended to spend 10 to 18 percent of their tax-cut income, they reported actually spending 28 to 43 percent of the funds. This is higher than estimates from studies of recent tax cuts, and arguably a consequence of the design of the 2011 tax cuts. The shift to greater consumption than intended is largely unexplained by presentbias or unanticipated shocks, and is likely a consequence of mental accounting. We also use data from a complementary survey to understand the heterogeneous tax-cut response

Initiation and propagation of mechanically induced neuronal injury

Traumatic brain injury (TBI) is an important but poorly understood cause of lifelong disability in the United States. Progress has been made in animate models of TBI, showing a clear relationship between the type and magnitude of injury and the histologic and behavioral outcomes, it is still not clear whether TBI-related pathologies are independent of similar stroke-related pathologies. The brain is generally considered protected from mechanical forces. However, during traumatic events, cells in the brain tissue are exposed to a complex set of mechanical forces that include transient acceleration, pressure, and direct stretch. This dissertation presents a model to expose cultured cells of the central nervous system (CNS) to a defined stretch insult and to monitor both the acute and delayed response of neurons to the stretch. The system is designed to apply a controlled, transient mechanical stretch to cultured cells, ranging from physiologic to traumatic. A fraction of the culture was deformed, allowing examination of the response of either stretched or unstretched cells in the same culture. Together, this system allows us to monitor the biochemical progression of the mechanically-induced changes in the cultured CNS cells. Acute changes in [Ca2+]i caused by a rapid stretch of cultured hippocampal neurons showed increases in [Ca2+] i correlated with the level of mechanical insult, and that the magnitude of the peak calcium influx exceeded levels initiated by NMDA or glutamate exposure. Despite this acute increase in [Ca2+] i, cell viability 24 hours following stretch-injury was unaffected for all but the most severely stretch-injured cultures. Likewise, in adjacent unstretched neurons, increases in intracellular calcium were also found in the acute period after stretch, but no viability change was observed in these neurons 24 hours after stretch. Consistent with previous studies, inhibition of the cytosolic calcium transient in neurons by calcium free media or MK-801 resulted in protection from NMDA neurotoxicity. In contrast, these treatments did not improve viability following mechanical stretch-injury. These data suggest that an acute increase in [Ca2+]i may not be a primary modulator of neuron death following mechanical deformation of neurons

Validation of MicroRNA Biomarkers for Alzheimer’s Disease in Human Cerebrospinal Fluid

We previously discovered microRNAs (miRNAs) in cerebrospinal fluid (CSF) that differentiate Alzheimer’s disease (AD) patients from Controls. Here we examined the performance of 37 candidate AD miRNA biomarkers in a new and independent cohort of CSF from 47 AD patients and 71 Controls on custom TaqMan arrays. We employed a consensus ranking approach to provide an overall priority score for each miRNA, then used multimarker models to assess the relative contributions of the top-ranking miRNAs to differentiate AD from Controls. We assessed classification performance of the top-ranking miRNAs when combined with apolipoprotein E4 (APOE4) genotype status or CSF amyloid-β42 (Aβ42):total tau (T-tau) measures. We also assessed whether miRNAs that ranked higher as AD markers correlate with Mini-Mental State Examination (MMSE) scores. We show that of 37 miRNAs brought forth from the discovery study, 26 miRNAs remained viable as candidate biomarkers for AD in the validation study. We found that combinations of 6–7 miRNAs work better to identify AD than subsets of fewer miRNAs. Of 26 miRNAs that contribute most to the multimarker models, 14 have higher potential than the others to predict AD. Addition of these 14 miRNAs to APOE4 status or CSF Aβ42:T-tau measures significantly improved classification performance for AD. We further show that individual miRNAs that ranked higher as AD markers correlate more strongly with changes in MMSE scores. Our studies validate that a set of CSF miRNAs serve as biomarkers for AD, and support their advancement toward development as biomarkers in the clinical setting

Ischemic preconditioning regulates expression of microRNAs and a predicted target, MeCP2, in mouse cortex

Preconditioning describes the ischemic stimulus that triggers an endogenous, neuroprotective response that protects the brain during a subsequent severe ischemic injury, a phenomenon known as ‘tolerance'. Ischemic tolerance requires new protein synthesis, leads to genomic reprogramming of the brain's response to subsequent ischemia, and is transient. MicroRNAs (miRNAs) regulate posttranscriptional gene expression by exerting direct effects on messenger RNA (mRNA) translation. We examined miRNA expression in mouse cortex in response to preconditioning, ischemic injury, and tolerance. The results of our microarray analysis revealed that miRNA expression is consistently altered within each group, but that preconditioning was the foremost regulator of miRNAs. Our bioinformatic analysis results predicted that preconditioning-regulated miRNAs most prominently target mRNAs that encode transcriptional regulators; methyl-CpG binding protein 2 (MeCP2) was the most prominent target. No studies have linked MeCP2 to preconditioning or tolerance, yet miR-132, which regulates MeCP2 expression, is decreased in preconditioned cortex. Downregulation of miR-132 is consistent with our finding that preconditioning ischemia induces a rapid increase in MeCP2 protein, but not mRNA, in mouse cortex. These studies reveal that ischemic preconditioning regulates expression of miRNAs and their predicted targets in mouse brain cortex, and further suggest that miRNAs and MeCP2 could serve as effectors of ischemic preconditioning-induced tolerance

Adenosine kinase inhibition protects against cranial radiation-induced cognitive dysfunction

Clinical radiation therapy for the treatment of CNS cancers leads to unintended and debilitating impairments in cognition. Radiation-induced cognitive dysfunction is long lasting, however, the underlying molecular and cellular mechanisms are still not well established. Since ionizing radiation causes microglial and astroglial activation, we hypothesized that maladaptive changes in astrocyte function might be implicated in radiation-induced cognitive dysfunction. Among other gliotransmitters, astrocytes control the availability of adenosine, an endogenous neuroprotectant and modulator of cognition, via metabolic clearance through adenosine kinase (ADK). Adult rats exposed to cranial irradiation (10 Gy) showed significant declines in performance of hippocampal-dependent cognitive function tasks (novel place recognition, novel object recognition, and contextual fear conditioning) 1 month after exposure to ionizing radiation using a clinically relevant regimen. Irradiated rats spent less time exploring a novel place or object. Cranial irradiation also led to reduction in freezing behavior compared to controls in the fear conditioning task. Importantly, immunohistochemical analyses of irradiated brains showed significant elevation of ADK immunoreactivity in the hippocampus that was related to astrogliosis and increased expression of glial fibrillary acidic protein (GFAP). Conversely, rats treated with the ADK inhibitor 5-iodotubercidin (5-ITU, 3.1 mg/kg, i.p., for 6 days) prior to cranial irradiation showed significantly improved behavioral performance in all cognitive tasks 1 month post exposure. Treatment with 5-ITU attenuated radiation-induced astrogliosis and elevated ADK immunoreactivity in the hippocampus. These results confirm an astrocyte-mediated mechanism where preservation of extracellular adenosine can exert neuroprotection also against radiation-induced pathology. These innovative findings link radiation-induced changes in cognition and CNS functionality to altered purine metabolism and astrogliosis, thereby linking the importance of adenosine homeostasis in the brain to radiation injury